The complex events that occur during the replication of eucaryotic genomes are not yet understood. By analogy with knowledge of prokaryotic systems, it is expected that much of the basic information about chromosomal replication might be obtained by studying the replication of the genomes of extrachromosomal elements and other viruses. The same analogy predicts that great progress in this area will be achieved by in vitro replication systems that carry out faithful replication of such genomes as in vivo replication. A low eucaryote yeast Saccharomyces cerevisiae has many advantages over other eucaryotes. The system is much less complex. It is very easy to prepare a large amount of cells and to do its enzymology. It has one of the best genetics and has the extrachromosomal element, 2-Mum plasmid, whose replication is the same as chromosomal replication. Furthermore, autonomously replicating sequences (ARSes) have been isolated from yeast chromosomes and are maintained extrachromosomally in yeast. An in vitro replication system of 2-Mum plasmid and ARS-1 DNA which mimics in vivo replication has been recently developed. The specific aims of this project are using yeast (1) to prove ARSes are in fact DNA replication origins in vivo, (2) to study how the DNA replication origins are temporarily ordered and their initiation is regulated, (3) isolation, mapping, and characterization of new temperature-sensitive DNA replication mutants, (4) cloning, overproduction, purification, and functional analyses of their gene products, (5) factionation and reconstitution of the in vitro replication system of yeast 2-Mum plasmid DNA. To this end, we will be able to understand how initiation of DNA replication is operated by various protein components, how such reaction is controlled, how DNA strands are elongated, how DNA replication is terminated, and how DNA strands are segregated.